1. Field of the Invention
The present invention relates generally to the use of novel S(N-hydroxycarbamoyl) glutathione mono- and di-ester derivatives as potent cytotoxic and anti-tumorigenic agents, and to a method of treating tumorigenic diseases with these derivatives. More specifically, this invention relates to the use of S-(N-hydroxycarbamoyl) glutathione mono- and di-ester derivatives, and salts thereof, in suppressing and inhibiting tumorous and metasiatic growth in humans.
2. Description of the Background
Cancer can develop in the tissues of any organ at any age. The etiology of cancer is not clearly defined but mechanisms such as genetic susceptibility, chromosome breakage disorders, viruses, environmental factors and inmmunologic disorders have all been linked to malignant cell growth and transformation.
Tumorigenic growths are a most serious threat in modern times. Malignant or cancerous growths produce uncontrolled cell proliferation which results in unregulated growth of malignant tissue, lack of differentiation and an ability to invade local and even remote tissues. These growths have become, due to their unique characteristics, some of the most serious diseases encountered in modern medicine. Unfortunately, during the development of diseases associated with neoplastic growth there has been a lack of detectable symptoms and, for the most part, no completely effective therapy and/or prevention.
The seriousness of most cancer diseases and the lack of satisfactory treatment is accompanied by extreme secondary side effects found to accompany all current forms of cancer therapy. The success of surgery, the most radical treatment, depends on the stage when the cancer growth is discovered. If a whole tumor is discovered and removed before metastases develop, then surgery may be effective. In the majority of cases, however, the cancer is discovered too late for surgery to be effective as the sole treatment. Moreover, other available therapies, such as radiotherapy and chemotherapy, are accompanied by severe adverse reactions. In the case of radiotherapy, the sublethal doses of radiation used still adversely affect non-tumor tissues of the patient.
Anti-neoplastic chemotherapy currently encompasses various groups of drugs. Alkylating agents that alkylate cell protein and nucleic acids disrupt cell replication and metabolism and lead to cell death. Typical alkylating agents are nitrogen mustard, cyclophosphamide and chlorambucil. These agents are highly toxic and produce nausea, vomiting, alopecia, hemorrhagic cystitis, pulmonary fibrosis and an increased risk of development of acute leukemia. Purine, pyrimidine and folate antagonists are cell cycle and phase specific. In order to exert their anti-tumor effect, cells must be in the cell replication cycle and in the DNA synthesis phase of replication. Purine antagonists such as 6-mercaptopurine or 6-thioguanidine inhibit de novo purine synthesis and interconversion of purines. Pyrimidine antagonists, such as cytarabine, 5-fluorouracil or floxuridine inhibit DNA synthesis by inhibiting deoxycytidylate kinase and DNA polymerase. Folate antagonists such as methotrexate bind tightly to the intracellular enzyme dihydrofolate reductase, ultimately causing cell death from the inability to synthesize pyrimidines. Toxicities associated with the use of these compounds include alopecia, myelosuppression, vomiting, nausea, and cerebellar ataxia, among others.
Plant alkaloids such as vincristine, vinblastine or podophyllotoxins etoposide and teniposide generally inhibit mitosis and DNA synthesis and RNA dependent protein synthesis. The toxicities of these drugs are similar to those described above and include myopathy, myelosuppression, peripheral neuropathy, vomiting, nausea and alopecia.
Anti-tumor antibiotics such as doxorubicin, daunorubicin and dactinomycin act as DNA intercalators, preventing cell replication, and inhibiting the synthesis of DNA-dependent RNA and DNA polymerase. Bleomycin causes the scission of DNA and mitomycin acts as an inhibitor of DNA synthesis by bifunctional alkylation. These antibiotics are extremely toxic and produce necrosis, myelosuppression, anaphylactic reactions, anorexia, dose-dependent cardiotoxicity and pulmonary fibrosis.
Other compounds used for the chemotherapy of cancer are inorganic ions such as cisplatin and biologic response modifiers such as interferon, and various hormones. These compounds, similar to those mentioned above, are accompanied by toxic adverse reactions, and their use is limited due to severe side effects.
Most of the chemotherapeutic cancer treatments described above specifically target rapidly dividing cells by inhibiting DNA/protein synthesis. Unfortunately, rapidly dividing normal cells, like those of the intestinal epithelium and bone marrow, are also adversely affected by these drugs. This accounts for the severe side effects associated with cancer chemotherapy. Recent studies by the inventors and others suggest that the glyoxalase pathway plays a critically important detoxification role in cells. The quantitative differences in the levels of the glyoxalase enzymes in normal tissues versus neoplastic tissues is the basis of the alternative chemotherapy of the invention.
The glyoxalase enzyme system is a simple metabolic pathway, composed of just two enzymes that function to chemically remove cytotoxic methylglyoxal from cells as D-lactate, as shown in Scheme 1: ##STR2##
Glyoxalase I (hereinafter referred to as "Glx I"), the first enzyme in the pathway, catalyzes the conversion of the thiohemiacetal formed by glutathione (GSH) and methylglyoxal (M) to S-D-lactylglutathione (P). Glyoxalase II (hereinafter referred to as "Glx II"), the second enzyme in the pathway, catalyzes the hydrolysis of S-D-lactoylglutathione (P) to D-lactate (L). Methylglyoxal has been shown to arise as an unavoidable by-product of the enzymatic and nonenzymatic isomerization of intracellular triosephosphates. Previous reports that formaldehyde dehydrogenase can efficiently use methylglyoxal as a substrate, thereby by-passing the glyoxalase pathway, appear to be erroneous. In support of a detoxification role for the glyoxalase pathway, a mutant strain of the yeast Saccharomyces cerevisiae, which is defective in Glx I, is eventually killed by exposure to glycerol due to the accumulation of intracellular methylglyoxal. Moreover, E. coli cells amplified with the gene for Glx I exhibit enhanced resistance to the growth-inhibitory effect of methylglyoxal. The substrate specificities and kinetic properties of the glyoxalase enzymes are consistent with a pathway whose purpose is to efficiently remove methylglyoxal from cells. A quantitative kinetic model for the conversion of methylglyoxal to D-lactate in erythrocytes has recently been formulated by the inventors that emphasizes the high level of kinetic efficiency of the pathway. The following two major conclusions emerge from this kinetic model.
a) The pathway overall is about 50% of maximal kinetic efficiency in the sense that the thiohemiacetal substrate partitions nearly equally between formation of D-lactate and specific-base catalyzed dissociation to form free methylglyoxal (M) and GSH. That is, the nonenzymatic rate of formation of the thiohemiacetal is significantly rate determining. PA1 b) Both of the "diffusion-controlled" glyoxalase enzymes, i.e., enzymes whose efficiency is dependent upon the rate at which the substrate diffuses to the enzyme, are about 50% of maximal efficiency in the sense that bound substrates partition nearly equally between product formation and dissociation from the surface of the enzyme. PA1 R' and R.sup.a, which may be the same or different, are hydroxyl, --O--(C.sub.1 -C.sub.18)alkyl, pharmaceutically-acceptable salts thereof, or mixtures thereof. PA1 R' and R.sup.a, which may be the same or different, are selected from the group consisting of hydroxyl and --O(C.sub.1 -C.sub.18)alkyl; pharmaceutically-acceptable salts thereof; and mixtures thereof.
Thus, the pathway appears, at least in normal cells, to have achieved "optimal" kinetic efficiency in the sense that pathway velocity is now constrained by two fundamental physicochemical constants of water. These are the biomolecular rate of diffusion-controlled encounter between substrates and active sites, and the autoprotolysis constant of water that controls the specific-base catalyzed formation/decomposition of the thiohemiacetal at physiological pH 7. Presumably, this high level of kinetic efficiency arose during the course of biological evolution because of the selective advantage of minimizing the steady-state concentration of methylglyoxal in cells. This implies that the inhibition of the glyoxalase pathway should have severe deleterious effects on the cell.
From a historical perspective, early interest in the glyoxalase pathway as an anti-cancer target was based on reports that methylglyoxal has anti-neoplastic activity when tested against a range of different tumor types including Ehrlich ascites carcinoma, Yoshida ascites sarcoma, Kirkman-Robbins hamster hepatoma, adenocarcinoma, lymphosarcoma, and leukemia L4946. This effect might be due to inhibiting protein synthesis at the level of translation.
Indeed, certain S-aryl glutathione derivatives, known to be potent competitive inhibitors of Glx I, were found to inhibit the growth of L1210 leukemia and KB cells in culture (Vince et al., J. Med. Chem., 14:35-37 (1971)). The particular derivatives and the results obtained therefrom are shown in Table 1.
TABLE 1 ______________________________________ Inhibition of Glyoxalase I and L1210 and KB Cell Cultures by Glutathione Derivatives (GSR) I.sub.50a LD.sub.50b No. R (mM) (L1210, mM) (KB, mM) ______________________________________ 1 (CH.sub.2).sub.2 COC.sub.6 H.sub.5 0.077 0.08 0.07 2 CH.sub.2 C.sub.6 H.sub.3 Br-P 0.009 &gt;0.20 0.11 3 C.sub.6 H.sub.2 (NO).sub.3.sup.-2,4,6 .sub.c 0.12 0.05 4 C.sub.6 H.sub.3 (NO.sub.3).sub.2.sup.-2,4 0.766 &gt;0.20 0.06 ______________________________________ .sub.a I.sub.50 = concentration of 50% inhibition of glyoxalase I. .sub.b Concentration of compound for 50% kill of the cell culture. .sub.c No inhibitory activity was detected.
Vince et al., however, disclosed neither a clear correlation between the extent of cell growth inhibition and the K.sub.i values of the inhibitors, nor any reason to believe that highly charged GSH derivatives could diffuse across cell membranes. Thus, the observed cell growth inhibition could not be clearly ascribed to inhibition of intracellular Glx I. Nor did Vince et al. suggest how inhibition of Glx I could form the basis of a tumor-selective anticancer strategy.
The present inventors thus sought to overcome the problems of inhibitor transport and tumor-selectivity by using carefully designed inhibitors of Glx I. For example, based on the observation of Meister and coworkers that GSH[glycyl]ethyl ester is more rapidly transported than GSH into cells (Anderson et al., Arch. Biochem. Biophys., 239:538-548 (1985)), the present inventors believed that derivatives of GSH might be delivered indirectly into cells as their ethyl esters. Once inside the cell, the ester will be catalytically hydrolyzed to give free GSH.
Regarding the Glx II enzyme of the glyoxalase pathway, it is known in the art that several different types of cancer cells contain reduced levels of glyoxalase II activity in comparison with normal cells (Jerzykowski et al., Experientia (Basel) 31:32-33 (1975); Jerzykowski et al., Int. J. Biochem. 9:853-860 (1978); and Thornalley, Biochem. J. 269:1-11 (1990). For example, a comparison of the activity of Glx I and Glx II in normal and cancer cells is set forth in Table 2, below.
TABLE 2 ______________________________________ Some reported examples of glyoxalase activity in normal cells versus cancer cells Glyoxalase (mU/mg protein) Tissue I II GlxI/GLxII ______________________________________ Normal Cells: Erythrocytes 129 .+-. 32 50 .+-. 13 2.6 (human).sup.a Brain 1113 .+-. 19 817 .+-. 156 1.4 (human).sup.a Liver 209 .+-. 56 360 .+-. 13 0.6 (human).sup.a Heart 339 .+-. 24 280 .+-. 47 1.2 (hamster).sup.a Kidney 323 .+-. 48 330 .+-. 86 1.0 (human).sup.a Tumor cells: Leukemia 1210 113 .+-. 56 -0 large (mouse).sup.a Glioblastoma 290 .+-. 56 53 .+-. 10 5.5 (human).sup.a Kirkman-Robbins 540 .+-. 48 40 .+-. 10 13.5 hepatoma (hamster).sup.a Fibroadenoma 419 .+-. 73 27 .+-. 7 15.5 mammae (human).sup.a Bladder HT1197 542 .+-. 38 8 .+-. 1 67.8 (human).sup.b Kidney OUR10 321 .+-. 86 44 .+-. 16 7.3 (human).sup.b Prostate PC3 4206 .+-. 294 45 .+-. 3 93.4 (human).sup.b Testis T1 4767 .+-. 275 94 .+-. 12 51.0 (human).sup.b Colon HT29 542 .+-. 59 11 .+-. 1 49.3 (human).sup.b ______________________________________ .sup.a Jerzylowski, et al., Int. J. Biochem. 9:853 (1978). .sup.b Ayoub, et al., Anticancer Res. 13:151 (1993).
In view of the above, there thus exists a need in the art for GSH derivatives which are sufficiently hydrophobic to transport across the cell membrane into cells, for example, as their alkyl esters, and which will be subject to intracellular hydrolysis to produce free GSH.
In addition, there is a need in the art for compounds which will selectively inhibit or retard the growth and proliferation of neoplastic cells without affecting normal cells in vivo. In this regard, there is a need in the art for competitive inhibitors of Glx I that also serve as substrates for Glx II. It is believed that such compounds would function as tumor-selective anti-cancer agents, based on the observation that Glx II activity is abnormally low in some types of tumor cells. These inhibitors could, therefore, induce higher steady state concentrations of cytotoxic methylglyoxal in tumor cells than in normal cells because of the reduced ability of tumor cells to hydrolyze the inhibitor.
There is also a need in the art for pharmaceutical compositions useful for inhibiting or retarding the growth and proliferation of neoplastic cells and/or a tumor in a mammal which is not susceptible to multi-drug resistance. Multi-drug resistance is due to decreased accumulation of drugs in cells caused by increased drug efflux and/or decreased cell permeability mediated by the multi-drug transporter protein. This will occur for hydrophobic natural products, semi-synthetic analogs of such products, and synthetic organic compounds which are amphipathic and preferentially soluble in lipid. There thus exists a need in the art for pharmaceutical compositions comprising compounds to which multi-drug resistant cells are not resistant.
Finally, there is a need in the art for a novel method of inhibiting or preventing the growth of neoplastic cells and/or a tumor in a mammal. A method which selectively inhibits or prevents the growth of neoplastic cells and/or a tumor without inhibiting or preventing the growth of normal cells would be desirable in the art.